The proposed work is aimed at unraveling the molecular mechanism by which telomeres prevent the activation of DNA damage signaling pathways at chromosome ends. The loss of telomere protection and the resulting signaling by DNA damage response pathways is the root cause of the premature aging symptoms in Dyskeratosis congenita and other telomeropathies. Furthermore, telomere dysfunction is a key factor in the early stages of cancer, a disease strongly correlated with aging. Our work in the past funding period has illuminated how the telomere associated shelterin complex blocks the activation of the ATM and ATR kinase signaling cascades at chromosome ends. Using genetically modified mouse embryo fibroblasts (MEFs) with conditional alleles for the seven mouse shelterin factors, we have determined which shelterin subunits are required for the repression of the ATM and ATR pathways. This work has revealed a remarkable division of labor within shelterin where TRF2 is primarily responsible for the repression of ATM signaling whereas the POT1 proteins repress the ATR pathway. Using super-resolution STORM imaging in combination with a new chromatin spreading method, we have provided evidence that TRF2 represses the ATM kinase pathway through remodeling telomeres into the t-loop configuration. We have also provided evidence that the POT1 proteins repress ATR signaling through the exclusion of RPA, the single-stranded DNA sensor of the ATR pathway. In AIMs 1 and 2 of the current proposal, we will further test these two models. We propose to use our extensive collection of genetically modified MEFs in combination with STORM imaging, specifically engineered shelterin rescuing alleles, cell biological tests for the DNA damage response, and biochemical approaches with purified shelterin to test the t-loop model for ATM repression and the RPA-exclusion model for ATR repression. This work will be complemented with TALENs-mediated deletion of shelterin proteins in human cells to verify that data obtained with our mouse models are consistent with the human setting. In AIM 3, we will initiate a new line of investigation to determine how shelterin represses a third threat to telomeres - the activation of the poly(ADP-ribose)polymerases (PARPs) that can sense single- and double-stranded DNA breaks. Our recent data established that PARP1 is activated at dysfunctional telomeres, raising the question of how this powerful and potentially dangerous enzyme activity is normally repressed at functional telomeres. These experiments are designed to reveal the fundamental principles of telomere function in human and mouse cells with the objective to understand how loss of telomere function affects human health and aging diseases.